Our long-term objectives are to characterize the cis- and trans-acting factors that regulate initiation of replication in mammalian chromosomes, and to understand how chromosome architecture and transcription influence the activity of origins. Utilizing in vivo labelling and two-dimensional (2-D) gel replicon mapping methods, we have extensively characterized the dihydrofolate reductase (DHFR) replicon in Chinese hamster cells. Our data indicate that nascent DNA strands can initiate at any of a large number of sites scattered throughout the 55 kb intergenic region, but probably more often near the ori-beta and ori-gamma loci. However, results of a lagging strand assay suggest that more than 80% of initiations in the DHFR replicon occur within a 500 bp region centered over ori-beta. In addition, we have shown that deletion of the promoter of the DHFR gene, which lies 26 kb upstream from the nearest initiation site in the intergenic region, turns off origin activity in the intergenic region in the early S period. Our working hypotheses are: 1) that initiation in the DHFR locus is mediated by trans-acting factors that interact with cis-regulatory elements centered in ori-beta and ori-gamma to effect melting of the helix beginning at these sites; 2) that nascent strands can initiate at positions as far away as 10-15 kb on either side of these elements, but slightly more often near the elements themselves; and 3) that the activation of ori-beta and ori-gamma depends upon localized stress imparted through transcription of the DHFR and/or 2BE2121 genes in the early S period. Specific aims are as follows: 1) to determine why the 2-D gel methods and the lagging strand assay paint such different pictures of initiation in the DHFR locus; the lagging strand assay will be examined for potential artifacts, and a novel 2-D gel method will be used to ask whether there is a futile initiation cycle; 2) to identify any potential cis-regulatory elements in the DHFR initiation locus that are required for origin function; a series of cell lines will be created in which individual fragments from the initiation locus have been deleted by homologous recombination, and origin activity will then be assessed on 2-D gels; 3) to determine whether transcription of the DHFR or 2BE2121 genes potentiates origin activity in the intergenic region; gene replacement technology will be utilized to insert regulatable promoters into the single DHFR gene in a hemizygous cell line, and origin activity will be assessed in the presence or absence of transcription; and 4) to identify any alterations in chromatin fine structure in the initiation locus in response to origin activation or inhibition, using DMS-induced DNA/protein cross-linking, chemical DNA methylation high resolution micrococcal nuclease digestion, and in vivo footprinting; these studies may allow us to identify trans-acting factors that interact with specific cis- regulatory elements required for origin function in this locus.